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W R Fleischmann

Publications and source records attributed to W R Fleischmann.

At least 37 records · Page 2Linked to original sources

The interferons. Mechanisms of action and clinical applications.

The interferons (IFN) are one of the body's natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (alpha, beta, and gamma), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposi's sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs alpha and beta and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones.

Humans↗

Modulation of peripheral leukocyte counts in mice by oral administration of interferons.

The ability of interferons (IFN) to exert a systemic effect following their oral administration was evaluated. One systemic effect of parenteral interferon administration has been shown to be a suppression of the number of peripheral white blood cells both in man and in mouse models. Using the mouse model of peripheral white blood cell suppression, the relative systemic effects of orally and subcutaneously administered interferons were determined. Murine IFN-beta, murine IFN-gamma and cross-reactive recombinant human IFN-alpha A/D were examined. The oral administrations of each of the three interferons were found to cause a dose-dependent suppression of the peripheral white blood cell counts. Significant levels of suppression were seen with as little as 5 units/day of murine IFN-beta and with 500 units/day of recombinant human IFN-alpha A/D and murine IFN-gamma. The dose-response curves obtained with orally administered interferons were much more shallow than those obtained with subcutaneously administered interferons. The results demonstrate that oral administration of interferons can provide a significant systemic effect. Further, the results support the possibility that the oral administration of interferons may have therapeutic potential.

Administration, Oral↗

A simple and rapid method to determine hematopoietic growth factor activity.

A rapid and simple colorimetric microassay method for the determination of hematopoietic growth factor activities was established. The assay was used to detect CSF-1, GM-CSF, and IL-3 activities. The assay was based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by metabolically active cells. Results obtained with the colorimetric microassay are comparable with those obtained with the soft agarose assay. Advantages of the colorimetric microassay include the conservation of reagents, the shorter incubation time for the experiment, the shorter assay time, and the ability to evaluate large numbers of samples.

Animals↗

Effect of murine alpha-, beta-, and gamma-interferons in combination with alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, on the tumor growth and metastasis of B16 melanoma and Lewis lung carcinoma in mice.

We have previously established that type I interferon (IFN), a mixture of alpha- and beta-IFN, augments the antitumor activity of alpha-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, against B16 melanoma. The objective of the present investigation was to extend these earlier observations to metastatic Lewis lung carcinoma and to determine specifically which component(s) of type I IFN potentiates the antitumor activity of DFMO. Furthermore, we wanted to determine whether type II (gamma) IFN can also potentiate the antitumor activity of DFMO. Treatment of animals bearing Lewis lung carcinoma with DFMO, 2% in drinking water (3 g/kg/day), or IFN-alpha/beta (1000 units/mouse) given subcutaneously on alternate days for a total of ten doses alone resulted in 42 and 5% inhibition of tumor growth, respectively. A combination of DFMO and interferon brought about complete elimination of tumors in 12 of the 18 animals, and 94% inhibition of tumor growth in the remainder. DFMO or type I IFN administered alone caused 94 and 26% inhibition of metastasis, respectively. Combination treatment with these two agents resulted in complete elimination of visible metastases. Treatment of mice bearing B16 melanoma with DFMO resulted in 81% inhibition of tumor growth compared to controls. The administration of interferons alone resulted in tumor growth inhibition of 15, 3, 1, and 43% for type I, alpha-, beta-, and gamma-interferons, respectively. Treatment of animals with combination of DFMO and various interferons resulted in inhibition of 94, 93, 86, and 94% of B16 tumor growth for type I, alpha-, beta-, and gamma-interferons, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Interleukin 2 inhibits in vitro granulocyte-macrophage colony formation.

We have previously shown that murine bone marrow cells cultured with interleukin 2 (IL-2) produce interferon-alpha/beta (MuIFN-alpha/beta) and that IFN-alpha/beta can suppress in vitro granulocyte-macrophage colony-forming cell formation (GM-CFC). In this study, IL-2 was directly assessed for its ability to inhibit in vitro granulocyte and/or macrophage colony-forming cell formation (GM-CFC/M-CFC). C57BL/6 bone marrow cells were cultured with different colony-stimulating factors (CSF), i.e., partially purified macrophage-CSF (M-CSF) or recombinant granulocyte and macrophage CSF (GM-CSF) in the presence or absence of different IL-2 preparations. Partially purified mouse IL-2 or recombinant human or mouse IL-2 (rHuIL-2 and rMuIL-2) totally inhibit GM-CFC and M-CFC formation at 7 days of culture. The level of inhibition mediated by IL-2 was concentration-dependent, with as little as 1 U/ml giving total inhibition of colony formation. The ability of IL-2 to inhibit colony formation was completely abolished by treatment with antisera to IL-2. MuIFN-alpha/beta and MuIFN-gamma appeared to play no role in IL-2-induced myelo-suppression in that addition of antisera to these IFN failed to block IL-2-induced suppression. Myelo-suppression mediated by IL-2 was independent of the concentration of CSF used in the bone marrow cultures. Suppression was also not dependent upon the initial presence of T cells or natural killer (NK) cells. Bone marrow cells depleted of Thy-1+, Lyt-1+, Lyt-2+, NK-1.1+, Asialo GM1+, or Qa-5+ cells were as susceptible to IL-2 induced suppression as untreated or complement-treated bone marrow cells. These results suggest that IL-2 may play an important role in regulating different aspects of hematopoiesis.

Animals↗

Interferon review.

Although IFN proteins were recognized first for their potent antiviral properties, it has now been established that they may profoundly affect other vital cellular functions. The IFNs are divided into three main classes, alpha, beta, and gamma, and are defined by their differences in amino acid sequences, physicochemical properties, and induction by different agents from different cell types. The inducing agents include viruses, bacteria, bacterial products, polymers, low molecular weight compounds, and antigens or mitogens. Studies on the mechanisms of action of IFNs have mainly been focused on their antiviral actions. However, many of the facts revealed by these studies are equally relevant for understanding other actions of IFN. IFNs are extremely potent, they interact with specific receptors, and they induce the expression of specific genes, the products of which mediate their various actions. There is almost a complete lack of knowledge of what happens between the interaction of IFN with its receptor and induction of new RNA synthesis. However, we are beginning to understand how some of the IFN-inducible enzymes impair viral replication. The discovery of the dsRNA-dependent enzymes has implications beyond the IFN system. It is quite possible that they are used for other physiologic regulatory systems as well. The identities and functions of many other IFN-inducible proteins remain to be elucidated. Principally, IFNs alpha and beta are cytokines in that they may be produced by the cellular components of the immune system and have immunoregulatory effects on the cells of the immune system. These effects include enhancement of surface structures such as histocompatibility antigens, pleiotropic hormone-like effects, and stimulation or inhibition of the activities of a number of different effector cells such as B cells, T cells, macrophages, and natural killing cells. IFN levels may be below detection and yet mediate important biologic functions. Perhaps the most interesting IFN subtype regarding immunoregulation is IFN gamma, which is a product of T lymphocytes. Few drugs have stimulated as much research interest or clinical promise as the IFNs. Clinical trials in patients have shown most promise in coryza, herpes virus infections, papilloma virus tumors, hairy cell leukemia, multiple myeloma, and renal cell carcinoma. IFN gamma employed alone and in combination with IFN alpha may dramatically increase IFN's activity. IFN treatment combined with chemotherapy also may give enhanced antitumor activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Interferon-induced cytolysis correlates with the degree of transformation of epidermal cells.

The cytolytic activity of interferon (IFN) was evaluated for its ability to distinguish between paired sets of nontumorigenic/tumorigenic epidermal cells (JB-1/JB-8; D1/D11a) as well as a set consisting of nonpromotable, promotable, and tumorigenic epidermal cell lines (JB-6 clones 30, 21, and RT101). The viability of the cell types was measured in a microassay using histoplates. IFN-gamma and IFN-alpha/beta, when employed singly, demonstrated an equivalent and limited effect on the viability of all cell lines. Treatment of nontumorigenic and nonpromotable cell lines with the combination of IFN-gamma + IFN-alpha/beta also caused only a limited effect on cell viability, whereas treatment of tumorigenic and promotable cell lines with the combination of IFNs caused a marked decrease in cell viability. Thus, the cytolytic effect of IFNs employed in combination but not singly was discriminatory between tumorigenic and nontumorigenic cells as well as between promotable and nonpromotable cells. Addition of cytotoxic effector cells was found to have a relatively moderate effect on the survival of target cells that were treated with IFN-gamma or IFN-alpha/beta separately. Addition of cytotoxic effectors to target cells treated with the IFNs in combination had a discriminatory effect. Nontumorigenic and nonpromotable cells were moderately affected; the tumorigenic and the promotable cells, however, were markedly affected, resulting in their complete (or nearly complete) eradication. The results demonstrate that combination IFN treatment: has a discriminatory cytolytic effect on tumorigenic and promotable cells in contrast to their nontumorigenic and nonpromotable counterparts; and when added to cytotoxic effector cells can completely eradicate tumorigenic and promotable cells while having a significantly lesser effect on nontumorigenic and nonpromotable cells.

Animals↗

In vivo myelosuppression by combination interferon treatment: antagonism of MuIFN-gamma and MuIFN-beta myelosuppressive effects.

Interferon treatment has been shown to cause myelosuppression in man and in a mouse model. Combinations of interferon-gamma (IFN-gamma) with either interferon-alpha (IFN-alpha) or interferon-beta (IFN-beta) cause the synergistic enhancement of interferons' antiviral, antiproliferative, antitumor, and immunoregulatory activities. Thus, combinations of MuIFN-beta and either natural or recombinant DNA-derived MuIFN-gamma were evaluated for their ability to cause the synergistic enhancement of interferon's myelosuppressive activity. The combinations of interferons were evaluated in vitro in bone-marrow colony-stimulating assays. They were seen to potentiate the in vitro myelosuppressive effect of the interferons. The combinations were evaluated for their in vivo myelosuppressive effect in mice. Treatment with the separate interferons caused a significant reduction in the number of circulating leukocytes, suggesting a potent myelosuppressive effect. However, treatment with the interferons in combination caused an antagonism and led to a myelosuppressive effect which was no greater than that of the interferons alone. The combinations of interferons were employed at concentrations which have been shown to provide substantial potentiation of the antitumor action of the interferons against B-16 melanoma. Thus, the data suggest that combination interferon therapy employing IFN-gamma together with either IFN-alpha or IFN-beta provide a potentiated antitumor activity without increasing the myelosuppressive side effect of the therapy.

Animals↗

Regulation of interferon-beta activity by fibroblast cells.

Exogenously administered interferons are rapidly cleared from the body. Several pharmacological mechanisms have been implicated in this clearance; however, they do not entirely explain the different clearance rates of the interferons. Cultured cells were studied for their ability to regulate interferon levels in vitro. Preparations of MuIFN-alpha, MuIFN-beta, and MuIFN-gamma were exposed to cells in culture and monitored for any loss in titer. MuIFN-beta titers were found to be significantly reduced following exposure to mouse L-929 fibroblast cells. The reduction of MuIFN-beta activity appeared to be specific for fibroblasts, since the reduction occurred following exposure to L-cells and to mouse embryo fibroblasts, but not to mouse reticuloendothelial cells. Moreover, the ability of the mouse fibroblast cells to reduce MuIFN-beta titers was blocked if the cells were pre-treated with actinomycin D, suggesting that de novo RNA synthesis was required. The titers of IFN-alpha and IFN-gamma were not reduced following exposure to either fibroblast or reticuloendothelial cells. Thus, the reduction of interferon titer by fibroblasts was IFN-beta specific. Similarly, HuIFN-beta titers were reduced following exposure to human fibroblasts. The ability of fibroblast cells to reduce IFN-beta titers was also found to be species-specific, since human fibroblast cells reduced the titer of HuIFN-beta but not MuIFN-beta while murine fibroblasts reduced the titer of MuIFN-beta but not HuIFN-beta. These results suggest that IFN-beta-treated fibroblasts specifically regulate their response to IFN-beta by reducing the titer of the IFN-beta activity.

Animals↗

Quantitation of in vivo potentiation resulting from combined interferon therapy: antitumor effect against B-16 melanoma in mice.

In this study, murine interferons (IFNs) were employed separately and in combination at subeffective and effective antitumor concentrations in a mouse B-16 melanoma system. Murine IFN-gamma (MuIFN-gamma) was demonstrated to be approximately 20 times more potent than MuIFN-alpha and MuIFN-beta in this system. Potentiation was observed with combinations of both subeffective and effective concentrations of MuIFN-gamma with MuIFN-alpha or MuIFN-beta. The level of potentiation was observed to increase from undetectable to two-fold to fourfold with three increasing IFN concentrations. Thirty percent of the mice treated for 14 days with 332 U/day MuIFN-gamma plus 7,500 U/day MuIFN-alpha survived apparently tumor-free for 100 days. Forty percent of the mice treated for 14 days with 332 U/day MuIFN-gamma plus 7,500 U/day MuIFN-beta survived apparently tumor-free for 100 days. All control mice died by day 41. The results are consistent with the suggestion that combination IFN therapy may have some use in the control of at least some tumors in humans.

Animals↗

Regulation of interferon-induced antiviral states: contrasting profiles of decay in response to the removal of MuIFN-alpha, MuIFN-beta and MuIFN-gamma.

The stabilities of the antiviral states induced by different types of murine interferons (IFNs) were compared to gain insight into possible differences in the modes of regulation of their respective antiviral states. Both type I (MuIFN-alpha and MuIFN-beta) and type II (MuIFN-gamma) IFNs were employed to establish antiviral states against mengovirus in mouse L-929 cells. At various times after IFN treatment, the IFNs were removed and the stability of the antiviral states was determined by single cycle mengovirus yield reduction experiments. The antiviral states induced by the two type I IFNs decayed significantly by 12 h following IFN removal. The rate of this decay was an exponential function of the level of the antiviral state induced. A transcriptional block effectively delayed the decay of the antiviral state, suggesting the involvement of a positive feedback mechanism of regulation. In contrast, the profile of the antiviral state induced by type II IFN showed a significant enhancement upon MuIFN-gamma removal. This enhancement was not dependent upon de novo transcriptional and translational activity of the cells. These data suggest that the modes of regulation of the antiviral states against mengovirus induced by type I and type II IFNs are distinctly different.

Animals↗

Macrophage colony stimulating factor (CSF-1) blocks the myeloid suppressive but not the antiviral or antiproliferative activities of murine alpha, beta, and gamma interferons in vitro.

Colony stimulating factors have been shown to antagonize the bone marrow suppressive effects of interferons in vitro. The effect of partially purified murine macrophage colony stimulating factor (CSF-1) was evaluated for its ability to antagonize interferon's bone marrow suppressive effect and was measured for its effect against two other interferon activities. The effect of CSF-1 against interferon's bone marrow suppressive effect was measured in a bone marrow colony growth assay. The effect of CSF-1 against interferon's antiviral activity was measured in single-cycle vesicular stomatitis virus growth experiments. The effect of CSF-1 against interferon's antiproliferative activity was measured in 3-day cell growth kinetics assays using B-16 melanoma cells and J-774 reticulum sarcoma cells of macrophage origin. Each of the three types of interferon (MuIFN-alpha, MuIFN-beta, and MuIFN-gamma) were employed and had common effects, though they differed in the levels of their effective concentrations. Concomitant treatment with CSF-1 and interferon blocked the interferon mediated bone marrow suppression in a dose dependent manner but had no effect on the antiviral or antiproliferative activities of the interferons. Importantly, CSF-1 did not affect interferon's antiproliferative activity against the growth of J-774 reticulum sarcoma cells, even though CSF-1 is a macrophage colony stimulating factor and J-774 cells are of macrophage origin. The results suggest that colony stimulating factors might be employed to specifically protect bone marrow function during interferon therapy while not interfering with interferon's antiviral and antitumor activities.

Animals↗

Effect of hyperthermia on the antiproliferative activities of murine alpha-, beta-, and gamma-interferon: differential enhancement of murine gamma-interferon.

Fever is frequently an important side effect of interferon (IFN) therapy. Studies have shown that culturing interferon-treated cells at elevated temperature heightens the antiproliferative activity of IFN-alpha and IFN-beta. Since IFN-gamma has also been shown to be a potent antiproliferative agent, the effect of elevated temperature on IFN-gamma activity was compared to its effect on IFN-alpha and IFN-beta. Mouse B-16 melanoma cells were simultaneously cultured under cloning conditions at a range of temperatures (37.3, 38.1, 38.6, and 39.4 degrees C) in the presence of MuIFN-alpha, MuIFN-beta, and MuIFN-gamma. The antiproliferative activities of all three interferons were enhanced by incubation at the elevated temperatures. However, the elevated temperatures had a more dramatic enhancing effect on the antiproliferative activity of MuIFN-gamma (10-fold enhancement) than of either MuIFN-alpha or MuIFN-beta (2.9- and 3.4-fold enhancement, respectively). Next, the enhancing effect of elevated temperature (39.4 degrees C) was examined for a range of interferon concentrations. The degree of the enhancing effect increased with increasing concentrations of MuIFN-gamma but not with increasing concentrations of MuIFN-alpha or MuIFN-beta. Enhancing effects of temperature as high as 14-fold were observed for 100 units of MuIFN-gamma/ml. This dramatic enhancement was observed for both natural and recombinant MuIFN-gamma and was neither a function of greater relative perception of MuIFN-gamma titer at elevated temperature nor a function of greater relative stability of MuIFN-gamma at the elevated temperature. The differential enhancement of MuIFN-gamma activity by elevated temperature appeared to be specific for the antiproliferative activity, since the antiviral activity of MuIFN-gamma was not relatively more enhanced at 39.4 degrees C than were the antiviral activities of MuIFN-alpha and MuIFN-beta. These results suggest that fever may be an important factor in maximizing the antitumor effects of MuIFN-gamma and perhaps of human IFN-gamma. They also raise the possibility that a combination treatment regimen of hyperthermia and interferon therapy, particularly IFN-gamma therapy, may provide a significant antitumor effect.

Animals↗

Effect of hyperthermia on combination interferon treatment: enhancement of the antiproliferative activity against murine B-16 melanoma.

Previous studies have evaluated the effects of hyperthermia on the antiproliferative activity of interferon. The activities of all three types of interferon have been shown to be synergistically enhanced by hyperthermic conditions. Further, the antiproliferative activity of interferon has been shown to be synergistically enhanced by combinations of gamma-plus alpha- or beta-interferon. The question remained whether combining these two methods of enhancing interferon activity would lead to an even higher level of enhancement of antiproliferative activity or to an antagonism of their separate effects. To address this question, mouse B-16 melanoma cells were cloned at 37.3 degrees C and at 39.4 degrees C in the presence of various combinations of murine alpha/beta- and gamma-interferon. Potentiation of interferon's antiproliferative activity by combination interferon treatment was found to occur at both temperatures. Moreover, the level of potentiation was synergistically enhanced by hyperthermic conditions. The results suggest that a combined treatment regimen of hyperthermia and combination interferon therapy (gamma- plus alpha- or beta-interferon) may provide a highly potent antitumor effect.

Animals↗

Treatment of mice with macrophage colony stimulating factor (CSF-1) prevents the in vivo myelosuppression induced by murine alpha, beta, and gamma interferons.

Interferon (IFN) treatment has been shown to suppress the bone marrow in mice and humans, causing a leukopenia. In vitro studies with the mouse system have shown that colony stimulating factors (CSFs) antagonize the bone marrow suppressive, but not the antiviral or antiproliferative activities of IFNs. Partially purified murine macrophage colony stimulating factor (CSF-1) was evaluated for its ability to antagonize the myelosuppressive effect of murine (Mu) IFNs in vivo. All three interferon types (MuIFN-alpha, MuIFN-beta, and MuIFN-gamma) were used. IFNs and CSF-1 were administered subcutaneously for 3 successive days, blood was drawn by retro-ocular bleeding, and myelo-suppression was quantitated by subjecting the blood to total white blood cell (WBC) counts and to differential counts. The IFNs had similar effects, though MuIFN-gamma was a significantly more potent (50- to 100-fold) myelosuppressive agent than MuIFN-alpha or MuIFN-beta. Treatment with each of the three IFNs caused a reduction in the total WBC count (up to 40% reduction) and a generalized reduction of each of the three major leukocytes (lymphocytes, segmented polymorphonuclear leukocytes, and monocytes). Treatment with CSF-1 did not affect the total WBC count, but did cause a shift in the relative ratios of the leukocytes, decreasing the relative percentage of lymphocytes, and increasing the relative percentage of segmented polymorphonuclear leukocytes and monocytes. Combined treatment with CSF-1 and IFN inhibited the myelosuppressive activity of the IFNs, and restored the total WBC count to control levels. This restoration was largely due to increased levels of segmented polymorphonuclear leukocytes and monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

An inhibitor of interferon action: II. Biological properties of the IFN-gamma-associated inhibitor of interferon action.

Previously an inhibitor of interferon action had been isolated from mitogen stimulated mouse spleen cells. This inhibitor was associated with the IFN-gamma molecule and might possibly represent an altered IFN-gamma molecule which can no longer induce an effective antiviral state. This report further investigates the biological properties of this inhibitor. Inhibitor activity is independent of the virus used in the interferon assay. Inhibitor activity has also been found to be species specific. Mouse inhibitor does not inhibit the antiviral activity of either human IFN-gamma or human IFN-beta. However, inhibitor production is not limited to the mouse system. Inhibitor is also present in preparations of human IFN-gamma. The inhibitor does not appear to directly compete with IFN-gamma for a specific cell surface receptor since inhibitor activity is independent of interferon concentration. The presence of inhibitor allows a unique, albeit reduced level of antiviral protection to develop. Increasing concentrations of interferon do not increase the level of antiviral protection allowed by the inhibitor. This inhibitor of interferon action may represent a natural mechanism whereby IFN-gamma-induced effects are regulated in vivo.

Animals↗